The mass spectrometry core will provide proteomics and metabolomics resources to enable the three major P01 projects achieve success in uncovering the molecular mechanisms of Hamartoma syndromes and related cancers in the TSC1-TSC2 pathways for new drug targets and novel therapies. For proteomics, microcapillary tandem mass spectrometry (LC/MS/MS) services will include protein complex identification, post-translational modification (PTM) site mapping such as phosphorylation, ubiquitination, acetylation, etc. and the relative and absolute quantification of peptides/proteins including modified peptides using both stable isotope labeling (SILAC, ITRAQ, TMT) and label-free quantification [spectral counting, total ion current (TIC), multiple reaction monitoring (MRM)]. These studies will be performed from cell lines, xenografts in addition to in vivo tissue sources from mice and human tumors. For metabolomics, services will include polar metabolite profiling using selected reaction monitoring (SRM). We will profile cells, tumor tissues and biological fluids using both steady-state profiling and stable isotope labeled C glucose/glutamine and N flux experiments to determine which metabolic pathways are altered in cells harboring defects in the TSC pathways. For these studies, the core will use a Thermo Fisher Scientific hybrid linear Ion trap-Orbitrap XL-ETD mass spectrometer and an AB/SCIEX 5500 hybrid QTRAP triple quadrupole mass spectrometer. For phosphorylation studies, we will use a combination of CID and ETD fragmentation. The majority of these studies will take place from immunopurified (IP) protein complexes in the relevant TSC1-TSC2 pathways and from phosphopeptide enrichment with IMAC and TiOa. Using both hybrid ion trap-orbitrap mass spectrometry via spectral counting and average TIC in addition to triple quadrupole mass spectrometry via MRM, we will develop quantitative clinical assays that will aid in the mechanistic deduction of pathway activation. The core will further develop in-house software to improve our informatics infrastructure necessary to analyze the data from protein-protein interaction (PPI) and quantitative PTM studies. We will also utilize the drosophila PPI dataset from -300 bait-prey IP-LC/MS/MS experiments from 36 proteins in the insulin signaling pathway from the first granting period by overlapping the IP-MS data via reciprocal BLAST with mammalian PPI bait-prey datasets in the TSC1-TSC1 pathway to identify novel protein pathway members followed by biochemical validation for functional significance.